DESCRIPTION(Adapted from applicant's abstract): The serotonin transporter (SERT) belongs to a large super family of closely related transporters, each of which couples the uptake of a neurotrasnmitter or biactive amine to the transmembrane gradients of Na+ and Cl-. SERT and its close relatives the dopamine and norepinephrine transorters are the molecular targets of antidepressants, cocaine and amphetamines in the brain. Analysis of their primary sequences suggests that these transporters contain 12 hydrophobic membrane-spanning domains, connected by hydrophilic loops of varying lengths. Although this general topology is supported by experimental evidence, very little other information is available about the three-dimensional structure or mechanism of SERT. Such information would be a critical step towards understanding the molecular details of drug biniding, in turn perhaps leading to the rational design of new therapeutic agents. A major portion of the proposed work will concern the detailed investigation of a large, previously made collection of mutatns in transmembrane span 7 of SERT. Studies will focus on the functional significance of a stripe of critically important residues, which may form part of the lining of a water-filled pore, or, alternatively, may form an important contact region with other membrane spans. Experiments using these mutants will also investigate span 7's nearest neighbors in SERT's three-dimensional structure, and the physical basis and functional significance of an apparent interaction between span 7 and extra cellular loop 1. Later in the project period, site-directed mutagenesis will be used to make a similarly comprehensive collection of mutatns for another membrane span in SERT, which will also be used to investigate SERT structure and function. These studies will provide important molecular details about SERT's mechanism that are likely to apply to the many other structurally related, physiologically important transporters in this super family.